The body usually completely metabolizes fats to carbon dioxide and water. However, if an inadequate amount of carbohydrate is present in the diet, or if a defect in carbohydrate metabolism or absorption is present, the body then metabolizes increasing amounts of fatty acids When large amounts of fatty acids are metabolized, fatty acid utilization is incomplete. Therefore, the intermediate products of fat metabolism appear in the blood and are excreted in the urine. These intermediate products are termed ketone bodies; and include acetoacetic acid, acetone, and .beta.-hydroxybutyric acid. In addition, stress, physical exercise and diabetes can cause an accelerated decomposition of fats and oxidation of fatty acids to increase the concentration of .beta.-hydroxybutyric acid, acetone and acetoacetic acid in the blood and urine. Accordingly, the assay for ketone bodies in a biological fluid can be helpful in the diagnosis, treatment and monitoring of diabetes.
The concentration of ketone bodies present in the urine and blood of a healthy individual is very low to negligible. Whenever increased amounts of fats are metabolized, such as when the carbohydrate intake is restricted or when the diet is rich in fat, the concentration of ketone bodies can increase. If an excess amount of ketone bodies is present in the blood, the condition is termed ketosis; and if an excess of ketone bodies if present in the urine, the condition is termed ketonuria. Ketonuria also is observed from the restricted carbohydrate intake that occurs with fevers, anorexia, gastrointestinal disturbances, fasting, starvation, cyclic vomiting, pernicious vomiting of pregnancy, cachexia, postanesthesia, and as a result of certain neurologic disorders. In general, all three ketone bodies are present in the urine of individuals with ketonuria in the relative proportions of 20% acetoacetic acid, 2% acetone, and 78% .beta.-hydroxybutyric acid. Acetone and .beta.-hydroxybutyric acid are derived from acetoacetic acid.
Diabetes mellitus is the most important disorder associated with ketosis or ketonuria. Diabetes mellitus is a disorder of glucose metabolism, and, in insulin-deficient diabetes, usually the juvenile-onset type, glucose metabolism is sufficiently impaired such that fatty acids are utilized to meet the energy requirements of the body. If diabetes mellitus is untreated, or is inadequately treated, excessive amounts of fatty acids are metabolized. Consequently, ketone bodies accumulate in the blood, i.e., ketosis, and are excreted in urine, i.e. ketonuria. In addition, ketone bodies are excreted from the body in combination with normal basic ions, thereby reducing in the carbon dioxide combining power of the body and causing systemic acidosis, i.e. increased acidity of the blood. Progressive diabetic ketosis causing diabetic acidosis can lead to coma, and eventually death. The term ketoacidosis is frequently used to designate the combined ketosis and acidosis conditions associated with diabetes.
Thus, detection of ketosis or ketonuria in an individual with diabetes mellitus is important and often indicates that a change in insulin dosage or other management procedures is necessary. Therefore, during periods of acute infections, surgery, gastrointestinal disturbances, or stress, and whenever the management routine does not adequately control the disease, the blood or urine of a diabetic individual should be checked for the presence of ketone bodies.
Usually, the presence of ketone bodies has been detected by assaying urine. In ketonuria, the acetoacetic acid, acetone and .beta.-hydroxybutyric acid are excreted in the urine. Consequently, an assay procedure that detects or measures the presence of one of the three ketone bodies usually is satisfactory for the diagnosis of ketonuria. Although specific tests exist for the determination of each of the ketone bodies, the specific tests usually are not used because the methods are more cumbersome, less reliable and less sensitive than the general assay for ketone bodies.
For example, the nitroprusside ion, (Fe(NO)(CB).sub.5).sup.-2, interacts both with acetone and acetoacetic acid in the presence of alkali to produce a purple-colored compound. Thus, sodium nitroprusside assays are specific to acetoacetic acid and acetone and do not detect .beta.-hydroxybutyric acid. This nitroprusside interaction forms the basis of a number of different prior art assays, such as Rothera's test and Legal's test. The present day reagent strip method is the simplest technique for determination of ketonuria The reagent strip is impregnated with sodium nitroprusside and alkaline buffers. The strip is dipped into fresh urine, then compared to the color chart after exactly 15 seconds. The chart has six color blocks indicating negative, trace (5 mg/dL), small (15 mg/dL), moderate (40 mg/dL), large (80 mg/dL), or (160 mg/dL) concentrations of ketones, and ranging in color from buff to lavender to maroon. However, the nitroprusside assay method does not measure the concentration of .beta.-hydroxybutyrate, the major ketone body. Accordingly, the assay result can be a misleading determination of the total amount of ketone bodies in the urine.
For example, studies have shown that assaying urine for acetoacetate by the nitroprusside method failed to detect ketonuria in from about 50% to about of diabetic individuals actually having ketonuria. Coupling this fact of misdiagnosis with the fact that assaying urine for ketones already is a delay in detecting blood ketosis, makes it obvious that a urine assay for acetoacetate is not sufficient to monitor the onset of ketosis in a diabetic individual. Accordingly, the present invention is directed to assaying a biological fluid, such as blood or urine, for the major ketone body, D-.beta.-hydroxybutyric acid, to achieve a sensitive and reliable assay for the onset of ketosis.
Although methods of determining the presence and amount of ketone bodies in urine are available, with advances in blood glucose self-monitoring by diabetic individuals, instances of undetected ketosis have increased. It has been found that as diabetic individuals increasingly use blood glucose monitoring to replace urine glucose testing, urine testing for ketone bodies then is neglected totally and ketonuria goes undetected.
D-.beta.-hydroxybutyric acid is present in the blood at about two to three time the level of acetoacetic acid. The presence of D-.beta.-hydroxybutyric acid in the blood also signifies the onset of ketosis much earlier than the detection of acetoacetare in the urine, and is a much more accurate analyte for monitoring the presence of ketone bodies, and hence, the effectiveness of a particular insulin therapy. For example, often, a sufficient amount of insulin is administered to drive down the glucose level of an individual, but not enough insulin is administered to drive down the level of ketone bodies. Furthermore, under some pathological conditions of the liver, all ketone bodies are converted to D-.beta.-hydroxybutyric acid. Therefore, an assay for acetoacetate could not detect such pathological conditions.
Accordingly, investigators sought methods of accurately detecting D-.beta.-hydroxybutyric acid in the blood. The most widely-used colorimetric test for D-.beta.-hydroxybutyric acid has been the reduction of the colorless dye 2-(4-indophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride hydrate to a colored formazan compound. However, this common dye is highly photosensitive, and is ascorbate and glutathione sensitive.
More particularly, the prior art, such as Japanese Patent Application Number 58-39813, filing date Mar. 8, 1983, disclosed assays for D-.beta.-hydroxybutyric acid (DHBA) wherein the DHBA is oxidized to acetoacetic acid by .beta.-hydroxybutyric acid dehydrogenase in the presence of nicotinamide adenine dinucleatide (NAD). This reaction produces reduced NAD (NAD-H), :hat in turn interacts with a tetrazolium dye to produce a colored formazan compound. The degree and intensity of the color transition then are correlated to the amount of DHBA in the test sample. In general, the reaction sequence is depicted as: ##STR1## Similarly, Harano et al., in the publication "Development of Paper-Strip Test for 3-Hydroxybutyrate and Its Clinical Application", Diabetes Care, 7, p. 481-485 (1984), disclosed a test strip assay method for DHBA, wherein the reaction sequence is similar to the sequence illustrated above in Equations (1) and (2). In addition, Harano et al., in the publication "Direct Automated Assay Method for Serum or Urine Levels of Ketone Bodies", Clinica Chimica Acta, 157, p. 177-183 (1985), disclosed an automated assay based upon the above reaction sequence.
European Patent Application No. 84306671.3 disclosed an assay for DHBA wherein the increasing amount of reduced NAD (NAD-H) generated in Equation (1) of the above sequence was determined photometrically. The assay included the addition of lactic dehydrogenase inhibitor, but did not include an indicator dye. Furthermore, the assay method was a wet assay method as opposed to a dry test strip assay method.
Owen, in U.S. Pat. Nos. 4,254,222 and 4,351,899, disclosed a method of assaying for DBHA based upon the above reaction sequence of Equations (1) and (2), but further included an intermediate electron carrier to more effectively reduce the tetrazolium dye to a formazan compound. Owen disclosed that improved color transitions were observed if the NAD-H reduced an electron carrier, like phenazine methosulfate, and then the reduced phenazine methosulfate reduced the tetrazolium dye to a formazan compound. As will be demonstrated more fully hereinafter, the composition and method of the present invention do not rely upon the tetrazolium reaction illustrated above in Equation 2. The present invention relies upon sequential enzyme-based reactions, and avoids the drawbacks and disadvantages associated with tetrazolium dyes, such as photosensitivity and sensitivity to ascorbate ion and glutathione often present in test samples
In contrast to the prior art, the method of the present invention utilizes a reductive pathway based upon lipoamide dehydrogenase (LADH) and a thiol-sensitive indicator dye, such as Ellman's reagent, a derivative of Ellman's reagent or, preferably, a substituted isobenzothiazolone. It has been found that, after the DHBA has reacted with DHBA dehydrogenase and NAD to form NAD-H, LADH then interacts with the NAD-H and D,L-lipoamide to form a thiol compound, 6,8-dimercaptooctamide. The 6,8-dimercaptooctamide then interacts with a thiol-responsive indicator dye, such as Ellman's reagent, a derivative of Ellman's reagent or a suitable isobenzothiazolone. Upon interaction with the thiol compound, the thiol-responsive indicator dye undergoes a detectable or measurable color transition that can be correlated to the amount of DHBA in a test sample.
As stated previously, the presence of ketone bodies in the blood or urine of an individual often is undetected because diabetics presently self-monitor glucose levels in their blood. The assay for ketone bodies is a standard urine assay, but since individuals started self-blood testing, the individuals avoid the bother and/or expense of buying and using a different assay product for use on a separate body fluid to test for another analyte. In addition, diabetics often believe, erroneously, that whole blood self-monitoring of glucose levels relieves the individual from the need to monitor ketones levels. However, if ketone monitoring is ignored, ketosis can go undetected, and the patient can slip into diabetic ketoacidosis, a common illness among patients with diabetes and having a mortality rate as high as 10%.
Therefore, uncontrolled diabetes has two important consequences. The first consequence is an alteration in glucose production and disposal, and the second is accelerated ketogenesis, i.e. ketone formation. These two consequences are closely connected because the metabolism of carbohydrates and lipids in the liver are tightly coupled, with elevated concentrations of acetoacetate and DHBA in the plasma of individuals with diabetic ketoacidosis due to accelerated synthesis of ketones in the liver and to the finite capacity of peripheral tissues to use these ketones.
Activation of ketogenesis requires the mobilization of long-chain fatty acids from stores in adipose tissue and a change in hepatic metabolism such that incomplete fatty acids are oxidized to ketone bodies rather than re-esterified to form triglycerides for transport out of the liver as very low-density lipoproteins. The mobilization of fatty acids is induced by insulin deficiency, whereas the oxidation of fatty acids is induced by a rise in glucagon/insulin ratio.
Ketone bodies then are metabolized by the body, with the ratio of DHBA to acetoacetate being extremely variable. However, the DHBA is the predominant component. The increasing levels of DHBA, as reflected in an increasing DHBA/acetoacetate ratio, correlates to the concentration of available free fatty acid. Since the rate of oxidation of the fatty acids is directly related to their plasma level, fatty acid concentration can determine the equilibrium between acetoacetate and .beta.-hydroxybutyrate. Therefore, a decreasing fatty acid oxidation can alter this equilibrium in favor of acetoacetate and result in a lower DHBA/acetoacetate ratio. Conversely, a very high DHBA/acetoacetate ratio is observed when fatty acid levels rise.
Insulin also has an immediate impact on the DHBA/acetoacetate ratio. A rapid decrease of the ratio occurs after administration of insulin. This effect is unrelated to the dose of insulin and is observed both in individuals with ketoacidosis and individuals attempting to stabilize of diabetes. The decrease of the DHBA/acetoacetate ratio is attributed to a delayed decrease in acetoacetate concentration, a concentration that can remain unchanged for hours and that can persist for days after commencement of ketoacidosis treatment. In fact, acetoacetate levels actually can increase during this period. Therefore, because the concentration of acetoacetate can increase, and at least remains unchanged, for several hours during ketoacidosis treatment, an assay for acetoacetate cannot reliably monitor the progress of the treatment.
Accordingly, the sensitive rise and fall of DHBA concentration at the onset of, and during treatment of, ketoacidosis signifies that DHBA should be the primary analyte for detecting and monitoring ketosis. For example, in terms of millimolar quantities, the extent of the rise and fall of blood levels of DHBA is greater than acetoacetate or glucose. Accordingly, an assay for DHBA provides the most sensitive monitor of ketosis and ketoacidosis among the various analytes that are symptomatic of ketosis.
Present-day technology has changed glucose self-monitoring from the rather inexact and post facto urine testing to the more sensitive metabolic control of blood glucose. Blood glucose monitoring is replacing urine testing since blood self-monitoring permits control of glycemia in the near-normal range. As a result, urinary ketone self-monitoring has been neglected. Apparently, the success of whole blood glucose self-monitoring has led individuals to neglect the monitoring of ketone levels in the blood or urine to signal the onset of ketosis. Therefore, because individuals and doctors have neglected testing for urine ketones, the development of a dry phase reagent test strip suitable to detect and measure blood ketone concentration would be useful for individuals monitoring an illness at home.
It has been demonstrated that any wet phase or dry phase assay that monitors only acetoacetate is unreliable as a monitor of the onset of ketosis. Therefore, due to the intimate interrelationship between glucose metabolism and ketone body metabolism in an individual with diabetes mellitus, it is apparent that the diabetic population that does whole blood glucose self-monitoring also needs a whole blood test for .beta.-hydroxybutyrate. As previously noted, the availability of test strips for blood glucose self-monitoring has increased the possibility of ketosis going undetected because individuals neglect urine testing, and the individual then fails to detect the onset of ketonuria. Thus, the availability of a dry phase reagent test strip for ketone bodies in blood would help prevent diabetic ketoacidosis. Further, if an individual because of preference or economics desires to monitor the onset of ketosis by urine testing for ketonuria, a sensitive and reliable urine assay to detect ketonuria should detect and measure DHBA. DHBA always is present in the urine in a greater concentration than acetoacetate, and therefore is the most sensitive and reliable analyte for the detection of ketonuria, and therefore, ketosis
Therefore, for an individual to detect the onset of ketosis or ketonuria, accurate and sensitive assays of whole blood, blood serum, blood plasma, urine and other test samples for ketone bodies are needed for both laboratory and home use. The assays should permit the detection and measurement of the ketone bodies in the test sample such that a correct diagnosis can be made and correct medical treatment implemented, monitored and maintained. In addition, it would be advantageous if the assay method utilizes a dry phase test strip, in either a wipe-off, a blot-off or a dip-and-read format, for the easy and economical, qualitative or quantitative determination of ketone bodies in blood, urine or other test samples.
Furthermore, any method of assaying for ketone bodies in blood, urine or other test sample should yield accurate, trustworthy and reproducible results by utilizing an indicator reagent composition that undergoes a color transition as a result of an interaction with a ketone body, like DHBA, and not as a result of a competing chemical or physical interaction, such as a preferential interaction with a test sample component other than a ketone body or a color transition occurring due to the instability of the indicator reagent composition. Moreover, it would be advantageous if the assay method for ketone bodies is suitable for use in dry phase reagent test strips for the rapid, economical and accurate determination of a ketone body in blood, urine or other test sample. Additionally, the method and composition utilized in the assay for ketone bodies should not adversely affect or interfere with the other test reagent pads that are present on a multideterminant reagent test strip.
Therefore, in order to determine if an individual is excreting ketone bodies or has an elevated amount of ketone bodies in the blood, and in order to monitor the course of medical treatment to determine the effectiveness of the treatment, simple, accurate and inexpensive detection assays for ketone bodies have been developed. Furthermore, of the several different assay methods developed for the detection or measurement of ketone bodies in urine, the methods based on dip-and-read dry phase test strips have proven especially useful because dry phase test strip methods are readily automated and provide reproducible and accurate results.
Some test strips used in assays for ketone bodies have a single test area consisting of a small square pad of a suitable carrier matrix impregnated with an indicator reagent composition comprising an indicator dye, such as a tetrazolium dye; NAD; and DHBA dehydrogenase. Other test strips are multideterminant reagent strips that include one test area for the assay of ketone bodies as described above, and further include several additional test areas on the same strip to permit the simultaneous assay of other test sample constituents. For both types of colorimetric test strips, the assay for ketone bodies is performed simply by contacting the colorimetric test strip with a blood or urine or other test sample, then comparing the resulting color of the test area of the test strip to a standardized color chart provided on the colorimetric test strip bottle, or determining the color change using a photometer. Ketone body tests usually are included on multideterminant reagent strips to screen urine samples during routine physical examinations. However, ketone body tests usually are not included on reagent strips used to assay blood samples.
The test strip method is the simplest and most accurate direct assay for the presence of ketone bodies in a biological fluid. In prior art test devices, the test area is impregnated with a tetrazolium indicator dye, NAD, and DHBA dehydrogenase. The test area transforms color when ketone bodies present in the test sample react with the DHBA dehydrogenase, NAD and tetrazolium dye in the test pad. In accordance with the above-described prior art method, an individual then can determine the concentration of a ketone bodies in a test sample from the color transition of the tetrazolium dye.
As will be discussed more fully hereinafter, investigators have found that tetrazolium dyes often are photosensitive and are subject to interfering interactions with common test sample components, such as glutathione and ascorbate, that substantially reduce the sensitivity and accuracy of the assay. Therefore, it would be extremely advantageous to have a simple, accurate and trustworthy method of assaying blood, urine and other biological fluids for ketone bodies. Present-day test strips for ketone bodies generally are not available for assaying blood and have the disadvantages of indicator dye instability and interference from test sample components. Surprisingly and unexpectedly, the composition and method of the present invention provide a stable indicator reagent composition that can be used in the assay of blood, urine or other biological fluids for ketone bodies. By providing a more accurate method of determining the concentration of ketone bodies in a test sample, in an easy to use format, such as a dip-and-read, a wipe-off or a blot-off test strip, the assay can be performed by laboratory personnel to afford immediate and trustworthy test results. In addition, the test strip method can be performed by the individual at home to more precisely monitor the level of ketone bodies in blood or urine and/or the success of the medical treatment the individual is undergoing.
The method of the present invention allows the fast, accurate and trustworthy assay for ketone bodies by utilizing a test strip that includes a test pad comprising a suitable carrier matrix impregnated with an indicator reagent composition of the present invention. The indicator reagent composition comprises a thiol-responsive indicator dye, such as Ellman's reagent, a derivative of Ellman's reagent or a substituted isobenzothiazolone; D-.beta.-hydroxybutyrate dehydrogenase (DHBA dehydrogenase); lipoamide dehydrogenase (LADH); D,L-lipoamide; and nicotinamide adenine dinucleotide (NAD). The indicator reagent composition is sensitive to low concentrations of the ketone body D-.beta.-hydroxybutyric acid (DHBA); is specific to DHBA; and essentially eliminates the problem of indicator dye and indicator reagent composition instability that leads to inaccurate and insensitive assays. Accordingly, the improved stability and selectivity of the indicator reagent composition enhance the sensitivity of the assay for DHBA, thereby providing a more accurate and trustworthy assay for ketone bodies.
Prior to the present invention, no known method of assaying blood, urine or other test samples for ketone bodies included an indicator reagent composition comprising a thiol-responsive indicator dye; DHBA dehydrogenase; LADH; D,L-lipoamide; and NAD to provide a stable and selective indicator reagent composition. Consequently, the improved stability and selectivity of the indicator reagent composition increase the sensitivity of the assay such that accurate and trustworthy assays for ketone bodies are achieved. Although a dry phase test strip including a tetrazolium indicator dye; NAD; and DHBA dehydrogenase has been used previously, dry phase test strips incorporating these compounds demonstrated an instability to light and a tendency to interact with various common test components. Accordingly, this instability and nonselectivity decreased the accuracy and the sensitivity of the test strip to the ketone bodies in the test sample.
In general, dry phase test strips are more advantageous than the wet phase assays because the test strip format is easier to use, requiring neither the continual preparation of reagents nor the attendant apparatus. In addition, reagent stability is greater in the test strip, thereby resulting in improved assay accuracy, sensitivity and economy. Notwithstanding that dry phase test strips for determining ketone bodies are more stable and more sensitive than wet phase assays, present day test strips for ketone bodies still need improvement in the areas of stability, selectivity and sensitivity. Therefore, it would be a significant advance in the art of diagnostic assays if test strips were even more stable during storage and even more selective and sensitive to ketone bodies. It was towards achieving these improvements that the investigations resulting in the composition, device and method of the present invention were directed.
Some attempts at achieving the above-mentioned goals of increased stability and sensitivity are found in the previously-discussed prior art. In contrast to the prior art, and in contrast to the presently available commercial test strips, the composition of the present invention imparts increased stability and improved selectivity to the test strip, and therefore increased sensitivity of the test strip, in the detection and measurement of ketone bodies in a test sample. The method of the present invention utilizes a stable indicator reagent composition that effectively resists degradation of the indicator dye and interacts specifically with the ketone body DHBA present in the test sample. Surprisingly and unexpectedly, the method and composition of the present invention essentially eliminate color formation, or other detectable responses, attributable to indicator dye interaction with test sample components other than DHBA. Hence, in accordance with the method of the present invention, new and unexpected results are achieved in the dry phase test strip assay of blood, urine and other test samples for ketone bodies by utilizing stable, selective and sensitive indicator reagent composition.